Background: Castrodia elata Blume (G. elata) is a traditional
Chinese herb used for centuries in folk medicine. Due to the claimed
anticonvulsant properties of C. elata, it is expected that this herb
continues to be a target of research, aiming to deepen the available
knowledge on its biological activity and safety. Purpose: The current
review aims to discuss the most recent advances on the elucidation of
the phytochemical composition and anticonvulsant potential of G. elata.

Methods: Available literature was reviewed from PubMed, ISI Web of
Knowledge and Science Direct, using combinations of the following
keywords: Castrodia elata, tianma, epilepsy, anticonvulsant and
pharmacokinetics. Abstracts and full texts were evaluated for their
clarity and scientific merit.

Results: G. elata rhizome, as well as specific phenolic compounds
isolated from this herb, have demonstrated anticonvulsant potential in a
variety of in vitro and in vivo models. The pharmacological mechanisms
potentially involved in the anticonvulsant activity have been
extensively studied, being similar to the known mechanisms claimed for
the available antiepileptic drugs. In addition, the pharmacokinetics of
the main bioactive component of G. elata (gastrodin) has also been
studied.

Conclusion: Due to its recognised therapeutic properties, C. elata
has gained an increasing interest within the scientific community and,
therefore, new medicinal preparations containing G. elata rhizome itself
or its bioactive components are expected to be developed in the coming
years. Moreover, specific phytochemical constituents isolated from G.
elata may also be considered to integrate programs of discovery and
development of new anticonvulsant drug candidates.

Keywords:

Epilepsy

Gastwdia elata

Gastrodin

Phenolic compounds

Anticonvulsant properties

Introduction

Natural products have been widely used as medicines, dietary
products and nutritional supplements since ancient times due to the fact
that they are a rich source of bioactive compounds and multiple benefits
for human health have also been shown. Effectively, the use of medicinal
plants has been validated by traditional use and they are time tested
when compared with modern medicinal herbal supplements (Atanasov et al.,
2015). For this reason, herbal plants and some of their bioactive
compounds have come to highlight their therapeutic potential and have
been the subject of extensive research (Kim et al., 2014), mainly at the
level of their biological activities and the underlying molecular
mechanisms of action. Consequently, natural products can represent
valuable starting materials for drug discovery programs (Bauer and
Bronstrup, 2014).

Gastrodia elata Blume (G. elata) is a traditional herb that has
been used in oriental countries for centuries (Kim et al., 2012a,
2003b). Its dry tuber is officially listed in the Chinese Pharmacopoeia
and has been used as an anticonvulsant, analgesic and sedative product
(Ojemann et al., 2006). However, it has been described as having a large
variety of other pharmacological properties. Additionally, several
phytochemical compounds isolated from this herb, such as gastrodin,
4-hydroxybenzyl alcohol (HBA), vanillin, vanillyl alcohol,
4-hydroxybenzaldehyde, [N.sup.6]-(4-hydroxybenzyl) adenine riboside
(NHBA) and parishins, have been proposed as playing an important role in
the pharmacological and therapeutic properties claimed for G. elata. The
majority of studies in this context has been focused on the potential
interest for central nervous system (CNS) disorders as it has been
recently reported (Jang et al., 2015). For instance, the treatment of
dopaminergic SH-SY5Y cells with an ethanol C. elata extract showed
protective effect on l-methyl-4-phenylpyridinium-induced cytotoxicity
(An et al., 2010) and similar results were observed with gastrodin Qiang
et al., 2014) and vanillyl alcohol (Kim et al., 2011). In in vivo
conditions, using a Parkinson's disease mouse model, an aqueous G.
elata extract revealed better antidyskinetic effects than those observed
with amantadine, a reference drug (Doo et al., 2014). Regarding
Alzheimer's disease, the ethyl ether fraction of a methanol G.
elata extract reduced the amyloid-[beta] peptide-induced cell death
similarly to melatonin (Kim et al., 2003), and an aqueous extract
improved cognitive functions in mouse and showed superior in vitro
results than the observed with Gouteng herb and huperzine A (Mishra et
al., 2011). Additionally, gastrodin decreased the area of amyloid-[beta]
peptide deposition in the cortex and hippocampus of Tg2576 transgenic
mice (Hu et al., 2014) and 4-hydroxybenzyl methyl ether exhibited
memory-ameliorating effects on SCH23390- and scopolamine-induced memory
impairment models (Lee et al., 2015). Considering the treatment of
cerebral ischemia, it has been reported an important neuroprotective
effect of vanillin and HBA against ischemic neuronal cell death in the
hippocampal CAI region in gerbils subject to transient global ischemia
(Kim et al., 2007). A long-term treatment of a rat model of
Tourette's syndrome with gastrodin also revealed promising results,
suggesting a dual restoring effect of gastrodin on the striatal dopamine
content (Zhang and Li, 2015). Concerning psychiatric disorders, an
aqueous extract of G. elata evidenced antidepressant-like effects
possibly via regulation of serotonergic and dopaminergic systems (Chen
et al., 2009) and this plant also showed potential benefits for the
treatment of schizophrenia in the phencyclidine mouse model (Shin et
al., 2010). An additional study also suggested anxiolytic-like effects
for HBA and 4-hydroxybenzaldehyde (Jung et al., 2006) and NHBA appeared
to potentiate the hypnotic effect of sodium pentobarbital in mice as an
agonist for both adenosine [A.sub.1] and [A.sub.2A] receptors (Zhang et
al., 2012).

Beyond the CNS effects of G. elata constituents, many other
pharmacological and therapeutic properties have been under
investigation. For example, an ethanol extract of G. elata (Ahn et al.,
2007) and vanillyl alcohol Uung et al., 2008) inhibited the angiogenesis
in the chick chorioallantoic membrane assay. Other G. elata extracts
similarly showed potential in alleviating tumorigenesis and exhibited
antimetastatic activity (Heo et al., 2007). In addition to its
anti-angiogenic effects, vanillyl alcohol also inhibited the vascular
permeability and exhibited anti-nociceptive activity probably through
the involvement of prostaglandin biosynthesis (Jung et al.. 2008).
Moreover, vanillin was found to be a potent anti-inflammatory and
analgesic compound, and 4-hydroxybenzaldehyde, HBA and benzyl alcohol
significantly inhibited the cyclooxygenase-1 and cydooxygenase-2
activities (Lee et al., 2006). The cardioprotective effects of G. elata
have also been studied, mainly focusing on blood pressure and serum
lipid levels, which were reduced by acidic polysaccharides purified from
Castrodia rhizomes in spontaneously hypertensive rats (Lee et al., 2012)
and Sprague Dawley rats (Kim et al., 2012b). A reduction in insulin
resistance was also observed in diet-induced obese rats treated with an
aqueous G. elata extract, and this effect was mainly attributed to the
phenolic compounds 4-hydroxybenzaldehyde and vanillin (Park et al.,
2011). Furthermore, gastrodin significantly prolonged the coagulation
time and decreased the fibrinogen content, suggesting its possible use
as a promising anticoagulant lead compound (Liu et al., 2006); gastrodin
also inhibited the cardiac hypertrophy induced by pressure overload in
mice (Shu et al., 2012). A methanol extract of G. elata also presented
gastroprotective effects (An et al., 2007) and, more recently, the
anti-osteoporosis activity of gastrodin has also been explored (Chen et
al., 2015a; Huang et al., 2015a).

The clinical evidence about the therapeutic properties of G. elata
and/or its phytochemical constituents is still scarce. However, a
double-blind, placebo-controlled clinical trial was already performed,
suggesting an anti-sickling effect of vanillin (Garcia et al., 2005).
Another double-blind, randomized, controlled clinical study was carried
out to investigate the preventive effects of gastrodin on the
neurocognitive decline, a common complication after cardiac surgery with
cardiopulmonary bypass; this study showed a significant decrease in the
neurocognitive decline in the patients treated with gastrodin (Zhang et
al., 2011). Moreover, a clinical study is ongoing to evaluate whether G.
elata is effective in the treatment of masked hypertension
(ClinicalTrials.gov, 2016).

Among the multi-pharmacological effects of G. elata and its
bioactive constituents on the CNS, the anticonvulsant effects are
certainly worthy of note (Ojemann et al., 2006). Indeed, epilepsy is one
of the most common serious neurological disorders, affecting around 60
million people worldwide (Shetty and Upadhya, 2016); moreover, despite
the large arsenal of antiepileptic drugs (AEDs) currently available,
approximately 30-40% of patients develop pharmacoresistance (Kwan et
al., 2010; Sorensen and Kokaia, 2013), thus existing an imperative need
of new AEDs with improved efficacy. Furthermore, over the years,
patients with epilepsy have used a variety of herbs to treat known
comorbidities of epilepsy and common adverse events of AEDs (Ekstein,
2015), and some medicinal plants have shown potential as new treatment
options for patients whose seizures are uncontrolled with the available
AEDs. Actually, extracts of plants and/or their single constituents have
shown to act on the same pharmacological targets as those of the most
commonly used AEDs (Sucher and Carles, 2015; Zhu et al., 2014).
Undoubtedly, the search for new anticonvulsant lead compounds among
phytochemicals constituents has emerged as a promissory and alternative
drug discovery approach. For instance, according to the progress report
on new AEDs published as a summary of the Twelfth Eilat Conference
(EILAT XII) that took place in Madrid (Spain) in 2014 (Bialer et al.,
2015), at least three of the AED candidates in clinical development are
herb-derived compounds (cannabidiol, cannabidivarin and huperzine A).
Therefore, taking into account the contribution of Ojemann and
collaborators (Ojemann et al., 2006) and the new literature available on
the anticonvulsant properties of G. elata extracts and some of their
phytochemical constituents, this review intends to address in a more
extensively manner the phytochemical composition of G. elata, as well as
discuss the more recent data on the pharmacokinetics and the scientific
evidence for potential therapeutic benefits in epilepsy.

Methods

To prepare this review, an extensive literature search from three
databases, PubMed, ISI Web of Knowledge and Science Direct, was
performed to generate a critical but comprehensive overview of the
phytochemistry, pharmacokinetics, pharmacological and anticonvulsant
properties exhibited by crude extracts or purified compounds of G.
elata. The keywords for the search consisted of combinations of the
following terms: Castrodia elata, tianma, epilepsy, anticonvulsant and
pharmacokinetics.

Botanical aspects

G. elata, commonly known as "Tianma" (Ojemann et al.,
2006), is a member of the Orchidaceae family (Tao et al., 2011).
Although the orchids usually are famous due to their beautiful flowers,
Gastrodia species have attracted attention because of their
pharmacological properties. Gastrodia plants, lacking green leaves and
chlorophyll, are saprophytic perennial herbs comprising approximately
twenty species worldwide. These species grow in the glades or at the
edge of forests in humid mountain areas at an altitude of about 400-3200
m, and the populations are isolated by lowlands with different
environmental conditions (Chen et al., 2014a). Specifically, G. elata is
found primarily in eastern Asia and in the mountainous ranges of China
and Korea (Ojemann et al., 2006). This species exhibits high level of
genetic diversity, which was mainly attributed to its perennial habit
and mixed reproduction system (Chen et al., 2011a). As this herb is
entirely dependent upon the fungus Armillaria mellea for nutriment
(Muszynska et al., 2011), the cultivation of this plant outside its
native region remains a challenge. In addition, because of its
over-collection as food and for medical nutrition therapy, it is
becoming increasingly rare in the wild state and, therefore, G. elata
has been currently listed as a rare and endangered plant species in
China and even in the world (Chen et al., 2011a). Thus, great efforts
towards its preservation based on genetic studies (Chen et al., 2014a,
2014b), as well as in new methods of cultivation have been under
investigation (Huang et al., 2011).

Phytochemistry

Over the years, several phytochemical studies have been mainly
focused on the isolation and identification of the phenolic elements of
G. elata. Indeed, phenolic compounds constitute the majority of the
total constituents of G. elata and they are usually considered
responsible for the main pharmacological and therapeutic properties
ascribed to this medicinal plant. This can be explained in part by the
fact that phenolic compounds are well recognized for their antioxidant
activity (Kancheva and Kasaikina, 2013), combating the reactive oxygen
species which, when produced in excess, are associated with several
physiological and pathological conditions (Rosenfeldt et al., 2013).
Therefore, several studies aiming at evaluating the antioxidant activity
of the compounds present in G. elata have been carried out, focusing
just on the antioxidant properties and/or assessing the impact of this
antioxidant activity in other health conditions in which oxidative
stress is suspected to play an important role, such as neurodegenerative
disorders (Hwang et al., 2009; Jung et al., 2007; Yu et al., 2005).
Overall, such compounds include simple phenols and phenolic conjugates
such as parishins. In summary, the compounds isolated from G. elata are
listed in Table 1.

Gastrodin--the main bioactive compound of G. elata

Gastrodin (p-hydroxymethylphenyl-[beta]-D-glucopyranoside) (Fig. 1)
is a simple phenolic glycoside and it was the first bioactive compound
isolated from C. elata (Ojemann et al., 2006). It is considered the main
and most important bioactive component extracted from this herb (Li et
al., 2001), having multiple beneficial properties. The importance of
gastrodin in the phytochemical composition of G. elata is highlighted by
the fact that the content of gastrodin is assayed as one of the most
important phytochemical markers in the quality standardization of G.
elata tubers (Tao et al., 2009).

Gastrodin is usually obtained by extraction from G. elata or by
chemical synthesis. Both of these methods are not straightforward, being
expensive and leading to many by-products and serious pollution problems
as well as wasting of natural resources. Thereby, new methods for the
synthesis of gastrodin have been developed over the years. In this
context. Zhu and collaborators reported the purification of the enzyme
responsible for the microbial transformation of 4-hydroxybenzaldehyde
into gastrodin from the fungal strain Rhizopus chinensis SAITO AS3.1165
(Zhu et al., 2010). In addition, the biotransformation of exogenous HBA
to gastrodin using hairy root cultures of Datura tatula L inoculated
with Agrobacterium rhizogenes is also a promising approach, with the
efficiency of HBA glycosylation reaching approximately 60% (Peng et al.,
2008). Using the same precursor, it was also demonstrated the production
of gastrodin by the fungi Penicillium cyclopium AS 3.4513 with a yield
of 65 mg/l (Fan et al., 2013).

General phenolic compounds

Although gastrodin has been considered the major active component
responsible for the medicinal properties of G. elata, diverse reports
have suggested that the pharmacological effects of this herb cannot only
be attributed to this compound alone (Yang et al., 2007). In fact, other
bioactive compounds can be isolated from this herb (Hayashi et al.,
2002) and can also be responsible for several bioactivities. In this
context, a highlight goes to HBA, a gastrodin metabolite, which is also
named gastrodigenin (Ojemann et al., 2006). The structures of these
compounds and of other main simple phenolic constituents that have been
isolated and whose pharmacological and therapeutic activities have been
studied are exhibited in Fig. 1.

Phenolic compounds containing a nucleoside

The NHBA (Fig. 2) is a prominent compound isolated from G. elata,
which has anxiolytic and sedative properties, among others. In fact,
several efforts have been developed towards the effective semisynthesis
of this compound (Huang et al., 2007) and to the study of its in vivo
metabolic pathways, identifying the major metabolites in rat urine and
plasma after oral administration (Lei et al., 2011). Additionally,
several NHBA derivatives were synthesized and evaluated for
Huntington's disease (Chen et al.. 2011b) and the 3-methoxy-NHBA
[[N.sup.6]-(3-methoxy-4hydroxybenzyl) adenine riboside] was also tested
for the treatment of insomnia (Shi et al., 2014).

Phenolic conjugates containing a citrate moiety

A group of phenolic conjugates containing a citrate moiety (Fig. 3)
has also been discovered in the composition of C. elata such as the
parishins and a citryl glycoside,
trimethylcitryl-[beta]-D-galactopyranoside, which is constituted by a
sugar unit identified as galactose and an aglycone moiety characterized
as a citric acid derivative (Choi and Lee, 2006). Interestingly,
parishins can function as a gastrodin source through their
metabolization after gastric absorption and, for this reason, they also
have to be considered in the studies that investigate the
pharmacological activities of this plant.

Polysaccharides

Other compounds isolated from the C. elata rhizome belong to the
group of polysaccharides and it was found that the presence of these
molecules can be related to the fact that the nutrition needed for C.
elata growth is mostly dependent on the fungus Armillaria mellea
(Muszynska et al., 2011). The polysaccharides isolated from this plant
consist of only glucose molecules and their structures are represented
in Fig. 4. Although we have not found reports of any polysaccharides
isolated from G. elata associated with anticonvulsant properties, they
have other biological activities, being mainly studied in the prevention
of cardiovascular risk (Lee et al., 2012; Ming et al., 2012). Also,
structureactivity relationship studies were carried out regarding their
anti-dengue virus bioactivity (Qiu et al., 2007) and anti-angiogenic
effects (Chen et al., 2012).

Pharmacokinetics

Up to date, a limited number of studies has been performed in order
to assess the absorption and biodisposition of G. elata constituents.
Even so, there are some non-clinical and clinical studies that reported
the pharmacokinetics of gastrodin and its metabolite HBA in in vivo
conditions. For instance, after intravenous (i.V.) gastrodin
administration (50mg/kg) to Sprague-Dawley rats, it was observed that
the parent compound (gastrodin) was rapidly distributed to the bile and
brain, and quickly biotransformed to HBA. In fact, HBA was found in the
bile and brain at 10 min post-dose and its levels rapidly declined after
gastrodin i.v. injection. The gastrodin in blood reached a peak
concentration of 24.1 [micro]g/ml whereas HBA attained a peak
concentration of approximately 220-fold lower (0.109 [micro]g/ml) at 15
min post-dose. The results of this pharmacokinetic study also suggested
that the brain exposure to gastrodin far exceeds the brain exposure to
HBA (more than 8.7-fold at 15 min after administration). Nevertheless,
taking into consideration the relationship between the systemic
concentration levels of both compounds, it can be inferred that the
metabolite HBA is able to pass through the blood-brain barrier in a more
efficient manner than gastrodin. Additionally, using microdialysis
probes, it was demonstrated that the recovery of both compounds
(gastrodin and HBA) in bile is higher than 90%; these findings suggested
the hepatobiliary system as the major route of gastrodin and HBA
excretion (Lin et al., 2008). According to the study of Lin et al.
(2007), the gastrodin brain-to-blood distribution ratio (k value) was
also found to be very low, being 0.007 [+ or -] 0.002 and 0.01 [+ or -]
0.002 at doses of 100mg/kg (i.v.) and 300mg/kg (i.v.), respectively.
Moreover, at this studied dose range (100 and 300mg/kg, i.V.), the
distribution and elimination processes of gastrodin in rat seem to
follow a linear kinetics (Lin et al., 2007). On the other hand, Wang et
al. (2008) also studied the gastrodin biodisposition in the rat after
the administration of 200 mg/kg (i.V.); the results obtained have also
shown that the entry of gastrodin into the brain is rapid, but the
extent of brain exposure was relatively small in comparison with the
extent of total systemic exposure, as assessed by the area under the
concentration-time curve (AUC). Indeed, the
[AUC.sub.brain]/[AUC.sub.plasma] ratios were not high; more
specifically, the individual ratios of the AUC in the cerebrospinal
fluid, frontal cortex, hippocampus, thalamus and cerebellum to the AUC
in the plasma were 4.8 [+ or -] 2.4%, 3.3 [+ or -] 1.2%, 3.0 [+ or -]
0.7%, 3.3 [+ or -] 1.3% and 6.1 [+ or -] 1.9%, respectively. As
demonstrated from these neuropharmacokinetic data, the cerebellum was
the brain region with a higher exposure to gastrodin, which may suggest
that this compound may have more potent effects on cerebellar targets
than in other brain areas. In this study, it was also shown that HBA is
immediately formed after i.v. gastrodin administration, but the measured
concentrations were very low and declined very quickly. Specifically,
the HBA concentrations were inferior to the lower limit of
quantification (LLOQ) of the bioanalytical method in plasma (LLOQ=0.15
[micro]g/ml) and in cerebrospinal fluid (LLOQ=0.07 [micro]g/ml) at 60
min and 90 min after dosing, respectively. Bearing in mind the
pharmacokinetic data provided by all these studies (Lin et al., 2008,
2007; Wang et al., 2008), it should be highlighted that gastrodin
undergoes a rapid biodistribution in rat, reaching quickly the CNS but
the concentration levels achieved therein are much lower than those
attained in blood. These findings suggest that the relatively small
amount of gastrodin that reaches the brain may be enough to elicit the
significant pharmacological effects in CNS ascribed to C. elata.
Nevertheless, more studies are necessary to better elucidate what is the
bioactive component that reaches the brain, as well as the contribution
of the main gastrodin metabolite (HBA) for the claimed therapeutic
properties. Additionally, after oral (p.o.) administration of different
preparations of C. elata to rats, the absorption of gastrodin was also
shown to be fast, with peak concentrations in plasma occurring at 10-20
min post-dose (Zheng et al., 2011).

The compound parishin, extracted from the roots of G. elata, was
also studied and, actually, this component can be considered a gastrodin
prodrug. In fact, after i.v. injection of parishin (116mg/kg) to Sprague
Dawley rats, the compound was converted in approximately 50% to
gastrodin, its main metabolite. The elimination half-life ([t.sub.1/2])
of parishin was low (0.29 [+ or -] 0.11 h), which suggests a fast
degradation and, interestingly, the gastrodin [t.sub.1/2] (1.17 [+ or -]
0.34 h) after the administration of parishin was similar to that
observed after the injection of gastrodin at 64.5mg/kg (1.31 [+ or -]
0.05 h) (Tang et al., 2015b). The same authors also intended to compare
the pharmacokinetic parameters of gastrodin, parishin and the ethyl
acetate fraction of ethanol extract of G. elata, considering that the
last two (parishin and G. elata extract) are gastrodin sources in vivo.
Interestingly, they discovered that these parameters are very different
depending on the administration route studied. In this study it was
found that the AUC (57.92 [+ or -] 11.94 [micro]g h/ml) of free
gastrodin was higher than the AUC of gastrodin originated from parishin
(4.96 [+ or -] 0.53 [micro]g h/ml) and from the G. elata extract (36.38
[+ or -] 3.84 [micro]g h/ml) and the peak plasma concentration
([C.sub.max]) was higher and had been reached earlier for free gastrodin
([C.sub.max] = 44.84 [+ or -] 14.51 [micro]g/ml at 0.42 h versus
[C.sub.max]=3.47 [+ or -] 1.58 [micro]g/ml at 0.83 h from parishin and
[C.sub.max] = 14.18 [+ or -] 3.94 [micro]g/ml at 1 h from the G. elata
extract) after intragastric administration. However, through the same
route the free gastrodin was faster eliminated, which was visible in its
[t.sub.1/2] (1.13 [+ or -] 0.06 h versus 3.09 [+ or -] 0.05 h from
parishin and 7.52 [+ or -] 1.28 h from the G. elata extract). A possible
explanation for this is the fact that parishins B and C, which already
are metabolites of parishin, can be further metabolized to gastrodin,
prolonging the in vivo gastrodin levels (Tang et al., 2015a). Indeed,
these results are very interesting, alerting to the point that the
pharmacological activities claimed for parishins in in vivo conditions
could actually be mediated by gastrodin or, ultimately, by HBA.

As it is believed that the main therapeutic actions of G. elata are
exerted in the CNS, the intranasal route can be a promising alternative
to traditional routes of administration due to the possibility to
circumvent the blood-brain barrier in the drug delivery to the brain. In
fact, intranasal gastrodin administration (50 mg/kg) provided an AUC in
the cerebrospinal fluid comparable to that obtained by i.v.
administration. Additionally, cerebrospinal fluid concentrations of
gastrodin 60 min following intranasal administration were always higher
than those achieved after i.v. administration, which suggested a direct
nose-to-brain pathway to transport gastrodin from the nasal cavity to
the brain (Wang et al., 2007). Later, a safe and stable in situ gel
preparation was formulated for the effective nasal delivery of gastrodin
in rats (Cai et al., 2011).

Undoubtedly, the data available on the pharmacokinetics of
gastrodin at the clinical level are still quite scarcer than those
generated from non-clinical conditions. Up to date, to the best of our
knowledge, the pharmacokinetic analysis of gastrodin was only considered
in one clinical study, which included eighteen male subjects to whom a
gastrodin capsule was orally administered at a dosage of 200 mg.
According to the obtained results, gastrodin was rapidly absorbed into
blood ([t.sub.1/2Ka] = 0.18 h) and reached a peak concentration at 0.81
h. In humans, the gastrodin concentrations achieved in plasma also
declined very rapidly, probably as a result of the fast distribution and
elimination processes (Ju et al., 2010), similarly to what was
previously referred in the rat.

Anticonvulsant properties and putative underlying mechanisms

As aforementioned, G. elata has been traditionally used to treat
epilepsy in oriental countries and its anticonvulsant properties have
been widely studied. Thus, in this section, the available scientific
evidence on the anticonvulsant activity of G. elata extracts and its
bioactive components, as well as their putative mechanisms of action, is
critically discussed. In addition, a summary of the main in vivo and in
vitro studies that support this activity is presented in Tables 2 and 3,
respectively.

G. elata rhizome extracts

The rhizomes of G. elata have been used to prepare different
aqueous and organic (e.g. methanol, ethanol) extracts (Yang et al.,
2007). Therefore, it is not surprising the reference to various G. elata
extracts (aqueous, methanol, ethanol), or even to fractions of such
extracts, in the multiple biological evaluation studies already carried
out (Heo et al., 2007; Park et al., 2011).

In general, the methanol extract of G. elata itself or different
fractions of this extract (n-butanol and ether fractions) appear to be
the most strongly associated with the anticonvulsant activity ascribed
to this herb. This can be related with the presence and amount of some
bioactive components in this type of extract, which may clearly depend
on the way the final G. elata herbal preparations are obtained (Yang et
al., 2007). In this context, Shin et al. (2011) observed that a methanol
extract of G. elata significantly delayed the seizure onset time and
shortened the seizure duration in a mouse model of acute cocaine-induced
seizures (Table 2, entry 1). A possible mechanism of action that could
explain such anticonvulsant activity was also explored in the same
study. Indeed, experiments showed that the anticonvulsant activity
mediated by the G. elata methanol extract was significantly reversed by
the [GABA.sub.A] receptor antagonist bicuculline (0.25 or 0.5 mg/kg,
i.p.) in a dose-related manner, but not by the [GABA.sub.B] receptor
antagonist SCH 50,911 (1.5 or 3.0 mg/kg, i.p.). These results suggest
that GABA receptors, particularly the [GABA.sub.A] receptor subtype, are
implicated in the G. elata-mediated anticonvulsant protection against
cocaine-induced seizures (Fig. 5). The same research group also
evaluated this G. elata extract in a chronic mouse model of epilepsy
induced by the administration of cocaine (15 mg/kg/day, i.p.) once every
2 days during 12 days, followed by 7 days of withdrawal before the
behavioural sensitization test; then, the methanol extract of G. elata
was given at 500 and 1000 mg/kg/day, p.o., during 20 days. Following
this experimental protocol, although the G. elata extract in both doses
did not attenuate the behavioural sensitization, it was able to block
the conditioned place preference induced by cocaine (Shin et al., 2011).
Additionally, a different mechanism was explored, being demonstrated
that the methanol extract of G. elata appeared to have neuroprotective
effects by reducing the toxicity induced by glutamate in HT22 cells
(Table 3, entry 1). These outcomes were proposed to be related with the
up-regulation of the phosphatidylinositol-3-kinase signalling pathway
and the antioxidant activity exhibited by this extract in the reduction
of the levels of reactive oxygen species induced by glutamate (Han et
al., 2014).

Regarding the anticonvulsant protection manifested by different
fractions of methanol extracts, it was found that the treatment with
repeated doses of an ether fraction of the methanol extract of G. elata,
during 14 days showed neuroprotective and anticonvulsant activity in the
kainic acid (KA)-treated mouse model (Fig. 5). Indeed, the ether
fraction of methanol extract of G. elata at the dose of 500 mg/kg
delayed the onset time of neurobehavioural changes and reduced the
severity of KA-induced seizures, as well as the hippocampal neuronal
damage in the CAI and CA3 regions (Table 2, entry 2) (Kim et al., 2001).
The same fraction was also evaluated in a subcutaneous
pentylenetetrazole (scPTZ)-seizure model. In this assay, scPTZ was
administered daily to rats, during 3 days, at a convulsive dose of 70
mg/kg. The recovery time was significantly reduced by the extract of G.
elata as well as the severity of the seizures, which was calculated by
the formula [SIGMA] [(degree of convulsion) x (frequency of convulsion
for each degree)]/total frequency of convulsion (Table 2, entry 3; Fig.
5). Then, in a chronic model of epilepsy consisting in the
administration of a subconvulsive dose (25 mg/kg) of scPTZ injected
during 8 weeks, the pre-treatment with the ether fraction of the
methanol G. elata extract lead to an increase on the GABA content levels
in the brain of scPTZ-treated rats (Table 2, entry 4) (Ha et al., 2000).
In another research work, among different G. elata studied extracts, the
n-butanol fraction of the methanol extract seems to be the most
effective in the inhibition of GABA-transaminase (GABA-T; Fig. 5), one
enzyme involved in GABA degradation (46.48 [+ or -] 5.24%) (Choi and
Lee, 2006).

Considering the KA-induced seizures in Sprague-Dawley rats, the
pre-treatment with an aqueous extract of G. elata also appeared to
reduce the number of three types of seizure attacks including wet dog
shakes, paw tremor, and facial myoclonus (Table 2, entry 5). In the same
study, it was demonstrated that the pretreatment with G. elata activated
the c-Jun N-terminal kinases signalling pathway and c-Jun expression. On
the other hand, the post-treatment with this herbal extract suppressed
both the c-Jun N-terminal kinases signalling pathway and the c-Jun
expression induced by KA (Table 2, entry 6). These results led the
authors to suggest that the modulation of activator protein 1 expression
via c-Jun N-terminal kinases signalling pathway by C. elata can
contribute to the anticonvulsant effect of this herb (Hsieh et al.,
2007). In order to understand the putative underlying mechanisms of
action of C. elata, Hsieh and collaborators also reported that an
ethanol extract of G. elata tested against neuronal damage in KA-treated
Sprague-Dawley rats indicated a reduction of microglia ac tivation and a
suppression of the neuronal nitric oxide synthase (Table 2, entry 7)
(Hsieh et al., 2005).

Gastrodin and HBA

Although the G. elata extracts have been frequently evaluated in a
range of different in vivo and in vitro assays, it is important to
remember that these herbal preparations consist in very complex mixtures
of bioactive components. Thus, with this kind of experiments, it is very
difficult to understand which bioconstituents are responsible for the
claimed pharmacological activities for the extracts. Therefore, as it is
known the phytochemical profile of the herbal extracts, it is common to
advance with the study of the properties of isolated biocompounds (e.g.
gastrodin). Due to the fact that gastrodin is considered the main
bioactive C. elata component, several studies have focused on the
evaluation of its biological activities either after purchase or
directly isolated from G. elata. Regarding its anticonvulsant
properties, gastrodin, isolated from the n-butanol fraction of a
methanol extract of C. elata, appeared to irreversibly inactivate the
succinic semialdehyde dehydrogenase (SSADH) of bovine brain in in vitro
conditions (Table 3, entry 2; Fig. 5) (Baek et al., 1999), thus
preventing the GABA degradation in the synaptic cleft. Later, a more
complete in vivo study evidenced that gastrodin isolated from the same
extract was able to decrease the immunoreactivities of the three enzymes
involved in GABA degradation (GABA-T, SSADH and succinic semialdehyde
reductase (SSAR)] (Table 2, entry 8; Fig. 5). The findings obtained in
this work also showed that the GABA-synthetic enzymes (two isoforms of
glutamic acid decarboxylase--GAD65 and GAD67) and GABA transporters are
not involved in the potential anticonvulsant mechanisms of action of
gastrodin. Additionally, gastrodin also reduced the seizure score in a
genetic seizure model (seizure-sensitive gerbils stimulated by vigorous
stroking of the back with a pencil) (Table 2, entry 8) (An et al.,
2003).

As it is widely recognised, monotherapy is considered the ideal
pharmacological approach in epilepsy treatment because of reduced side
effects, absence of drug interactions and better patient compliance.
However, due to the availability of multiple AEDs with different
mechanisms of action the possibility of "rational
polytherapy", taking advantage of possible synergism, is often an
option in patients with resistant epilepsy (Santulli et al., 2016).
Following this idea, a recent study was designed to evaluate the
anticonvulsant and neuroprotective effects resulting from the
co-administration of gastrodin and Phenytoin against seizures induced by
penicillin in mice; in the acute anticonvulsant experiments, both the
gastrodin alone ([ED.sub.50] = 950.60 mg/kg) and the Phenytoin alone
([ED.sub.50] =45.50 mg/kg) showed anticonvulsant activity, but it was
found that gastrodin and Phenytoin combination therapy can enhance the
anticonvulsant effect and reduce the side effects of Phenytoin; the
ideal phenytoin:gastrodin ratio was found to be 1:50 with an [ED.sub.50]
value of 8.59:429.27 mg/kg. This fact means that the dose of Phenytoin
was reduced by 81% and gastrodin by 55% without affecting the
anticonvulsant activity (Table 2, entry 9). Additional chronic
anticonvulsant experiments allowed assessing therapeutic and
neuroprotective actions, as well as side effects of the
phenytoin/gastrodin co-administration. As it is presented in Table 2
(entry 10), the combination therapy protected the normal balance and
memory function of the mice that were compromised by Phenytoin and
exhibited neuroprotective effects in the hippocampus (the neuron
morphology was preserved and the number of surviving neurons was higher
than the control group) (Zhou et al., 2015).

Although there are many studies reporting a wide spectrum of
biological activities of the gastrodin metabolite (HBA), in a
preliminary GABA-T inhibition assay it was found that this
bioconstituent isolated from the ether fraction of the methanol extract
of G. elata exhibited a very weak GABA-T inhibitory activity at a
concentration of 10 [micro]g/ml (Ha et al., 2001). Additionally, Choi
and Lee (2006) also evaluated HBA in the same assay (Table 3, entry try
3) (Choi and Lee, 2006) and, interestingly, the observed results confirm
its weak inhibitory activity against GABA-T previously reported by Ha et
al. (2001).

4-Hydroxybenzaldehyde and analogues

A study carried out by Ha and collaborators (Ha et al., 2001)
revealed that 4-hydroxybenzaldehyde, isolated from the ether fraction of
a methanol extract of G. elata, potently inhibited the activity of
GABA-T in in vitro conditions; a similar effect was exhibited by
4-hydroxybenzaldehyde analogues containing an aldehyde group attached to
the phenyl ring. The observed [IC.sub.50] values are present in Table 3
(entries 4-7), and it was clear that the inhibitory potency found for
these compounds on the activity of GABA-T (Fig. 5) is significantly
higher than that obtained for vigabatrin, a non-classic AED whose
mechanism of action is thought to involve the potentiation of GABAergic
neurotransmission by acting as an irreversible inhibitor of GABA-T
(Krasowski, 2010). Ha et al. (2001) also assessed the effect of these
compounds in the benzodiazepine receptors using a selective antagonist
([[sup.3]H]Ro15-1788) and a selective agonist ([sup.3]H]flunitrazepam).
The results showed that the compounds under evaluation (Table 3, entries
4-7) did not have agonistic activity on the [GABA.sub.A] receptor
complex (Ha et al., 2001). Indeed, 4-hydroxybenzaldehyde (5 x
[10.sup.-6]g/ml) had already shown a GABA-T inhibitory activity higher
than valproic acid (5 x [10.sup.-5]g/ml) (Ha et al., 2000). Furthermore,
structure-activity relationship analysis on 4-hydroxybenzaldehyde
derivatives as GABA-T and SSADH inhibitors indicated that a carbonyl or
an amino group as well as a hydroxyl group at the para-position of the
benzene rings are important for the inhibition of both enzymes. However,
the introduction of an alkyl group at the same position may reduce their
potency. Hence, the inhibition of these enzymes by the competitive
inhibitors 4-hydroxybenzylamine and 4-hydroxybenzaldehyde (Table 3,
entries 8 and 9) could result from the structural similarity between
both molecules and the two endogenous enzymes' substrates (GABA and
succinic acid), together with the conjugative effect of the benzene ring
(Fig. 5) (Tao et al., 2006).

Vanillin and vanillyl alcohol

In the evaluation of the anticonvulsant effects of vanillyl alcohol
on ferric chloride-induced seizures, it was observed that the
pre-treatment with this G. elata biocomponent was able to reduce the
counts of wet dog shakes, contrarily to the AED Phenytoin. Moreover, the
authors suggested that this effect could be related with the antioxidant
activity of vanillyl alcohol (200 mg/kg) in the rat brain, which was
stronger than the verified with Phenytoin, in both right and left brain
hemispheres (Table 2, entry 11) (Hsieh et al., 2000). Additionally,
vanillin can allosterically modulate the GABAergic neurotransmission by
enhancing the binding of the endogenous receptor agonist (Ha et al.,
2001). The glutamate-induced cellular apoptosis and the increase in
intracellular calcium induced by glutamate were also assessed in 1MR-32
human neuronal cells as possible anticonvulsant mechanisms of action of
vanillin and 4-hydroxybenzaldehyde (Table 3, entries 10 and 11).
Effectively, these G. elata constituents significantly inhibited both
the intracellular calcium rise and the apoptosis induced by the
excitatory neurotransmitter glutamate. In this study, similar results
were achieved when the cells were treated with EGTA (1 mM), an
extracellular calcium chelator (Lee et al., 1999); however, in this
study, it would also have been interesting to test as a possible
positive control a known AED acting as a calcium channel blocker and/or
interfering with glutamatergic system pathways. The vanillin
anticonvulsant activity was also evaluated through a model involving the
electrically induction of seizures (fully amygdala-kindled seizures), in
which the chronic state of epilepsy phenotype is established by repeated
application of sub-convulsive electric stimulation. In this model, the
pre-treatment with vanillin (administered intraperitoneally to rats 1 h
before stimulation), suppressed the stage 5 seizures with the median
effective dose ([ED.sub.50]) of 286 mg/kg; a similar result was achieved
with Phenytoin at 50 mg/kg (i.p.). Moreover, vanillin also induced a
significant shortened epileptic afterdischarge duration (Wu et al.,
1989).

Other bioactive constituents

The biocomponent trimethylcitryl-[beta]-D-galactopyranoside,
isolated from the n-butanol fraction of the methanol extract of G.
elata, was evaluated in the in vitro GABA-T assay. Although some degree
of GABA-T inhibition was evidenced by this compound (Table 3, entries
12; Fig. 5), it was not as effective as valproic acid (Choi and Lee,
2006).

In another study, the compound S-(4-hydroxybenzyl)glutathione,
isolated from an aqueous extract of G. elata, was tested in the
competitive binding assay with radiolabeled KA to the glutamate receptor
(Fig. 5), and it had a slightly lower affinity than the observed with
glutamate and glutathione (Table 3, entry 13) in the cortex of male
Wistar rats (Andersson et al., 1995).

Critical opinion

Nowadays, the majority of molecules tested as new AED candidates
follows the Anticonvulsant Screening Program of the National Institute
of Neurological Disorders and Stroke of the U.S. National Institutes of
Health. In fact, after its establishment in 1975, this program has made
relevant contributions regarding the development of new AEDs, such as
topiramate, lacosamide and retigabine, which were approved for epilepsy
(NINDS, 2016). Although this program is frequently used for the
identification of new chemical entities, it was surprising the scarcity
of information found for the extracts and/or biocomponents isolated from
G. elata in the first line of animal models usually used to predict the
anticonvulsant activity (maximal electroshock seizure test and scPTZ
test). In fact, there is only a research work that reports the
anticonvulsant activity of the ether fraction of methanol extract of G.
elata using an acute and chronic animal model of scPTZ (Ha et al.,
2000); however, the use of an electrical animal model of seizures (e.g.
maximal electroshock model or 6 Hz) was not found in the literature.

Even though several studies have focused on the mechanisms of
action (the so-called "advanced studies") of the extracts
and/or bioconstituents of G. elata, usually in research programs of
discovery of new anticonvulsant compounds these mechanistic assays are
considered at more advanced stages in order to explain the efficacy
and/or toxicity data previously obtained in animal models of seizures
(acute models) and epilepsy (chronic models). This is due to the fact
that the in vitro studies themselves do not give enough information
about the efficacy of the compounds as anticonvulsant drug candidates.
Therefore, further investigation is required to explore the effective
anticonvulsant potency of several of the discussed phytochemicals
contained in the G. elata. For instance, taking into account the strong
in vitro evidence highlighting the role of 4-hydroxybenzaldehyde in the
GABAergic system, an underlying putative mechanism of action of G. elata
against epilepsy (Ha et al., 2001, 2000; Tao et al., 2006), it is quite
surprising, at least to the best of our knowledge, that the
4-hydroxybenzaldehyde has not yet been investigated concerning its
anticonvulsant activity in acute seizure and/or epilepsy animal models.
In fact, 4-hydroxybenzaldehyde showed an inhibitory activity against
GABAT higher than that evidenced by HBA, the gastrodin metabolite (Choi
and Lee, 2006; Ha et al., 2001). In addition, integrating all the
aforementioned information on the anticonvulsant properties of G. elata
extracts and/or its biocomponents, it seems clear that there is a lack
of inclusion of positive controls (e.g. known AEDs) in the in vitro and
in vivo assays using seizures/epilepsy models, which would be useful to
better understand and clarify the anticonvulsant activity claimed for
the biocomponents existing in C. elata and/or the several G. elata
extracts.

Since the mechanisms of action of G. elata extracts and/or its
constituents have been widely explored, they are briefly illustrated in
Fig. 5. In this point, it is important to note that the two most
important neurotransmitters involved in the regulation of brain neuronal
activity are the excitatory neurotransmitter glutamate and the
inhibitory neurotransmitter CABA. In fact, changes in the ratio of
concentrations/activities of these compounds can contribute to increase
or decrease the predisposition for seizures (Rowley et al., 2012).
Therefore, several antiseizure mechanisms of action studied are oriented
towards the involvement of these neurotransmitters, particularly the
inhibition of enzymes involved in the degradation of GABA: GABA-T, SSADH
and SSAR. In addition to the research works described in this review,
although some other studies are not directly associated with
anticonvulsant activity, they also demonstrated the involvement of
glutamate and GABAergic systems in the action of several biocomponents
present in G. elata. In this context, a study conducted by Jung et al.
(2006) in mice demonstrated that 4-hydroxybenzaldehyde (100 mg/kg) can
interfere with GABAergic nervous system (Fig. 5) because its effects
were antagonized by flumazenil, a [GABA.sub.A] receptor antagonist (Jung
et al., 2006). More recently, the NHBA analogue
[N.sup.6]-(3-methoxy-4-hydroxybenzyl) adenine riboside (5 mg/kg)
increased the GABA levels in hypothalamus (39%) and cortex (32%) and
induced a significant decrease in glutamate contents in hypothalamus
(22%) and cortex (21%) of ICR mice. The authors suggested that these
effects could be associated with an increase in the glutamic acid
decarboxylase enzyme activity in hypothalamus (43%) and cortex (31%)
(Shi et al., 2014), which catalyses the decarboxylation of glutamate to
GABA (Fig. 5). In addition, immunohistochemical studies also showed that
NHBA (5 mg/kg, i.p.) increases the cFos expression in GABAergic neurons
of the ventrolateral preoptic area of Sprague-Dawley rats, which
suggests that NHBA activates the sleep centre in the anterior
hypothalamus, producing significant sedative and hypnotic effects (Zhang
et al., 2012).

Other molecular drug targets that also have a critical role in the
CNS disorders, including in epilepsy, are the ion channels. Actually,
the voltage-gated sodium channels are molecular targets for several
commonly used AED (e.g. Phenytoin, carbamazepine and lamotrigine) as
well as the calcium (e.g. gabapentin and ethosuximide) and potassium
(e.g. retigabine) channels (Loscher et al., 2013). Nevertheless, data
about the involvement of these ion channels in the putative
anticonvulsant/antiepileptic mechanism of action of G. elata
constituents remains scarce. In this regard, it was reported that
gastrodin regulates the potassium and sodium voltage-gated ion channels
in the small dorsal root ganglion neurons of diabetic rats (Sun et al.,
2012) and also inhibited proton-gated currents mediated by acid-sensing
ion channels in rat dorsal root ganglion neurons (Qiu et al., 2014).

The studies reported by Hsieh and collaborators (Hsieh et al.,
2007, 2005) should also be highlighted because effectively go further in
the research of potential epileptogenic processes than the classical
models commonly used to study the mechanisms of seizures for the
screening of new AED candidates. In fact, the complex mechanisms
underlying the epileptogenesis are misunderstood, leading to a higher
degree of difficulty concerning the emergence of new AEDs actually
efficacious in the epilepsy progression, which are able to prevent
refractory epilepsy as well as pharmacoresistance.

Since the AED candidates are intended to act in the CNS, it is
usual to evaluate in parallel their neurotoxicity and anticonvulsant
activity. In this context, the rotarod test seems to be the most used
assay to infer about the neuronal toxicity during the screening stages
of new AED candidates (Ghogare et al.. 2010; Hassan et al., 2012; Kumar
et al., 2011). In the rotarod assay, the side effects in the CNS are
manifested by the deficit in the motor coordination and this assay is
usually used to perform an initial screening of neurotoxicity and,
after, to calculate the median toxic dose ([TD.sub.50]), which allows
the estimation of the protective index ([TD.sub.50]/[ED.sub.50]) of the
compounds of interest. However, this test was not usually performed by
the authors who had assessed the anticonvulsant activity of G. elata
and/or its isolated constituents. An exception is the study conducted by
Descamps et al. (2009), in which the HBA was evaluated to determine the
minimal acute neurotoxicity through the rotarod assay, and at doses up
to 200 mg/kg no neurological deficit, such as ataxia and sedation, was
exhibited by C57 black J6 mice (Descamps et al., 2009).

Conclusion

The G. elata rhizome has been used as a traditional herbal medicine
for centuries. Several in vitro and in vivo studies have been performed
and many multi-pharmacological activities have been identified. However,
the most promising activities exhibited by this medicinal herb and/or
its phytochemical constituents are directed against several CNS
disorders, such as epilepsy. Due to the relevance of this plant over the
years, a more complete profile of the biochemical compounds isolated
from G. elata has been made and the study of the pharmacokinetics of
gastrodin (the most important phenolic compound) has brought an
increased knowledge about its possible CNS actions. Moreover, several
pharmacological mechanisms of action have been studied and proposed for
the anticonvulsant activity of either G. elata extracts or its
constituents and the available data appeared to be consistent and
reproducible. However, further investigation is required on this field.
Hence, more robust non-clinical studies and, in particular, clinical
trials are required, not only to further investigate the anticonvulsant
properties of G. elata extracts, but also to better understand what are
the constituents involved in the described pharmacological actions,
their mechanisms of action, toxicity profile and to confirm the claimed
therapeutic activity against epilepsy.

Conflicts of interest

The authors have declared no conflicts of interest. Acknowledgments

The authors are grateful to Fundacao para a Ciencia e a Tecnologia
(Lisbon, Portugal) for the PhD fellowship of Mariana Matias
(SFHR/BD/85279/2012), involving the POPH-QREN, which is co-funded by FSE
and MEC. The authors also acknowledge the support provided by FEDER
funds through the POCI--COMPETE 2020 --Operational Programme
Competitiveness and Internationalisation in Axis I--Strengthening
research, technological development and innovation (Project No. 007491)
and National Funds by FCT--Foundation for Science and Technology
(Project UID/Multi/00709). The authors also acknowledge the contribution
of Daniel Antunes Viegas for this review, particularly as
English-speaking qualified person.